Methods: Western Blotting is the multi step process to detect the protein present in the complex mixture. These Steps are as follows:

STEP 1: Preparation of Sample: Preparation of sample depends on the sample type.

For Tissue: For solid tissue such as tumor or whole liver, brain, it is first mechanically broken down into individual cells using a blender, homogenizer or by sonication. Once individual cells are obtained it will be processed as described.

For Cells: Individual cells are incubated with the lysis buffer containing detergent along with protease and phosphatase inhibitor cocktail to protect the sample from degration.

STEP 2: Electrophoresis of Sample: Sample was resolved on SDS-PAGE as described previously.

STEP 3: Transfer of protein gel on blotting membrane:

A. Preparation of Transfer membrane: Cut the membrane of the same size as gel.

A1: For nitrocellulose membrane: Place the membrane in the transfer buffer and observe that the liquid has wicked the membrane. Areas appeared as white spot needs special consideration.

A2: For PVDF membrane: Immerse the membrane into the 100% methanol for 15-30mins. Decant the methanol and submerge the membrane into the transfer buffer for additional 10-30mins.

B. Assembly of transfer cassette:

1. Remove the stacking gel from the PAGE and equiliberate the gl in transfer buffer for 10-30mins.

2. Place a pair of blotting sheet (already saturated with transfer buffer) on the anode plate (usually this plate is black colored).

3. Place the transfer membrane on the top of blotting sheets and remove trapped air bubble by rolling test tube or glass rod.

4. Place the PAGE gel on the top of membrane and gently remove trapped air bubble by rolling test tube or glass rod.